8 bromoadenosine 3 5 cyclic monophosphate camp

For the differentiation of both stromal-only and EO-only scaffolds, 10 nM β-oestradiol (E2, Sigma) was added 2 days post-cell seeding. For stromal cells only, at days 4 and 6, medium was replaced with the following conditions: 10 nM E2 + 1 μM progesterone (P4, Sigma) + 1 μM 8-bromoadenosine 3′,5′-cyclic monophosphate (cAMP, Sigma). For EO-only scaffolds, 20 ng ml⁻¹ prolactin (PRL, Peprotech, UK) was added in addition at days 4 and 6 to mimic signals from decidualized stromal cells. Supernatant was collected at days 2, 4, 6 and 8 and frozen at −20°C for storage before downstream analysis.
Enzyme-linked immunosorbent assays (ELISAs) were used to measure protein concentrations of key markers of stromal decidualization (prolactin, a hormone secreted by decidual stromal cells) and epithelial differentiation (glycodelin, one of the principal components of the endometrial glands) in the supernatant. ELISAs for prolactin (DY682, R&Dsystems, USA) and glycodelin (ELH-PP14, Raybiotech, USA) were measured from stromal-only and EO-only scaffolds, respectively. A volume of 100 μl of supernatant was used with each sample measured in duplicate, as per the manufacturer's instructions.

To assess the effects of chlamydial infection on the decidualisation of primary endometrial stromal cells, an in-vitro decidualisation method previously described was used^{32 (link)}. Cells were trypsinised, spun for 3.5 minutes at 800 rpm and resuspended phenol red-free RPMI 1640 with 10% CSFCS. They were then plated at 10⁵ cells per well of a 12-well plate for a minimum of 24 hours. Before the decidualisation protocol commenced, cells were serum starved in 2% CSFCS medium for 24 hours, supplemented as previously described. The cells were treated with 10–6 M progesterone (Sigma, Cat. No. P0130) and 8-Bromo-adenosine 3′-5′-cyclic monophosphate (cAMP, Sigma, Cat. No. A9501) at a final concentration of 0.1 mg/ml. The medium was changed every 48 hours over a six-day period (Supplementary Table S4).

Cryopreserved ME-derived SFCs (p1) were defrosted and plated in 24-well plates in SFC media; at confluence SFCs (p2) were incubated at 37 °C/5%CO₂ in decidualization media (2% FBS MSC-qualified, 1X glutamine, 100 units/ml penicillin-streptomycin, and 100μg/ml Normocin™ in Phenol Red Free DMEM with either 0.5 mM 8-Bromoadenosine 3′,5′-cyclic monophosphate (cAMP, Sigma-Aldrich, St. Louis, MO) ± 10 nM 17-beta estradiol (E2) (Tocris, Minneapolis, MN) and 1 μM medroxyprogesterone acetate (MPA, Sigma-Aldrich) or vehicle (PBS for studies with cAMP alone or 0.01% ethanol for studies with cAMP + E2 + MPA). Cells were stimulated for 6 h, 24 h, and 48 h, at which point supernatants and cell lysates were collected from each well for the time course experiment. Supernatants were analyzed for IGFBP-1 concentrations by ELISA (Duoset, R&D Systems, Minneapolis, MN) and results were normalized to cell lysate protein concentrations using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA).